Abstract
Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-C oV-2 ) is the causative agent of the COVID-1 9 pandemic
To explore the spatial and temporal aspects of SARS-C oV-2 replication at single-m olecule and cell levels, we carried out single-molecule fluorescence in situ hybridisation (smFISH) experiments with fluorescently labelled probes directed against the 30 kb viral genomic RNA (gRNA). 48 short antisense DNA oligonucleotide probes were designed to target the viral ORF1a and labelled with a single fluorescent dye to detect the positive sense gRNA, as described previously (Gaspar et al, 2017; Figure 1A)
We show that smFISH is a sensitive approach that allows the absolute quantification of SARS-C oV-2 RNAs at single-m olecule resolution
Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-C oV-2 ) is the causative agent of the COVID-1 9 pandemic. Fluorescence in situ hybridisation (FISH) was previously used to detect RNAs in hepatitis C virus and Sindbis virus-infected cells with high sensitivity (Garcia-M oreno et al, 2019; Ramanan et al, 2016; Singer et al, 2021) This approach has been applied to SARS-C oV-2 in a limited capacity (Burke et al, 2021; Rensen et al, 2021) with most studies utilising amplification-b ased signal detection methods to visualise viral RNA (Best Rocha et al, 2020; Carossino et al, 2020; Guerini-Rocco et al, 2020; Jiao et al, 2020; Kusmartseva et al, 2020; Lean et al, 2020; Liu et al, 2020). Our results uncover a previously unrecognised heterogeneity among cells in supporting SARS-C oV-2 replication and a surprisingly slower replication rate of the B.1.1.7 variant when compared to the early lineage VIC strain
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